Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay

TRPM8 (CMR1) is a Ca2+-permeable channel, which can be activated by low temperatures, menthol, eucalyptol and icilin. It belongs to the transient receptor potential (TRP) family, and therefore is related to vanilloid receptor type-1 (VR1, TRPV1). We tested whether substances which are structurally related to menthol, or which produce a cooling sensation, could activate TRPM8, and compared the responses of TRPM8 and VR1 to these ligands. The effects of 70 odorants and menthol-related substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells, were examined using a FLIPR® assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38, CoolactP and Cooling Agent 10) were found to be agonists. The EC50 values of the agonists defined their relative potencies: icilin (0.2±0.1 μM)>FrescolatML (3.3±1.5 μM) > WS-3 (3.7±1.7 μM) >(−)menthol (4.1±1.3 μM) >frescolatMAG (4.8±1.1 μM) > cooling agent 10 (6±2.2 μM) >(+)menthol (14.4±1.3 μM) > PMD38 (31±1.1 μM) > WS-23 (44±7.3 μM) > Coolact P (66±20 μM) > geraniol (5.9±1.6 mM) > linalool (6.7±2.0 mM) > eucalyptol (7.7±2.0 mM) > hydroxycitronellal (19.6±2.2 mM). Known VR1 antagonists (BCTC, thio-BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC50: 0.8±1.0, 3.5±1.1 and 18±1.1 μM, respectively). The Ca2+ response of hVR1-transfected HEK293 cells to the endogenous VR1 agonist N-arachidonoyl-dopamine was potentiated by low pH. In contrast, menthol- and icilin-activated TRPM8 currents were suppressed by low pH. In conclusion, in the present study, we identified 10 new agonists and three antagonists of TRPM8. We found that, in contrast to VR1, TRPM8 is inhibited rather than potentiated by protons. Keywords: TRPM8, CMR1, cold, menthol, VR1, odorants, proton activation, FLIPR, pain Introduction The recent cloning and characterization of the cold-menthol receptor (TRPM8; CMR1) (McKemy et al., 2002; Peier et al., 2002) was a major breakthrough in the study of thermosensation. TRPM8 is activated by menthol, eucalyptol and icilin, and by temperatures below ∼25°C. It belongs to the ‘long', or melastatin, subfamily of the transient receptor potential (TRP) family of ion channels (Montell et al., 2002), and shows pronounced outward rectification with a relatively high permeability for Ca2+ ions, and little selectivity between monovalent cations. The TRPM8 channel is expressed specifically in a subset of temperature-sensing trigeminal and dorsal root ganglion neurones (Peier et al., 2002; Reid et al., 2002a, 2002b; Nealen et al., 2003). Recently, a second cold receptor, ANKTM1, has been identified (Story et al., 2003), which, in contrast to TRPM8, is coexpressed with VR1 in a different subset of pain- and temperature-sensing trigeminal and dorsal root ganglion neurones. ANKTM1 is activated by icilin, but not menthol. These TRP channels play a major role in thermosensation (McKemy et al., 2002; Patapoutian et al., 2003). Although treatment with menthol or eucalyptol, or with cold temperatures, is a traditional method of pain relief (Wright, 1870; Green & Mcanliffe, 2000; Davies et al., 2002; Galeotti et al., 2002; Shanghai Medicinal Herbs, Essential Balm), little is known about the underlying analgesic mechanisms. It has been demonstrated that menthol blocks Na+ and Ca2+ channels in dorsal root ganglion cells (Swandulla et al., 1987; Haeseler et al., 2002). Others have postulated that the analgesic activity of (–)menthol is mediated by selective activation of κ-opioid receptors (Galeotti et al., 2002). The cold receptor TRPM8 is distantly related to the well-characterized heat-sensitive vanilloid receptor VR1 (or TRPV1). VR1 also belongs to the TRP channel family, but is activated by temperatures >42°C, or by ligands such as capsaicin and resiniferatoxin (RTX). Two endogenous VR1 agonists have been identified, anandamide (ANA) and N-arachidonoyl-dopamine (NADA) (Zygmunt et al., 1999; Di Marzo et al., 2001; Huang et al., 2002). Various VR1 antagonists have also been reported, for example, capsazepine, iodo-resiniferatoxin (I-RTX) and N-(4-tert.butyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydro-pyrazine-1(2H)-carboxamide (BCTC). These have analgesic effects in vivo (Bevan et al., 1992; Walpole et al., 1994; Catarina et al., 1997; 2000; Tominaga et al., 1998; Wahl et al., 2001; Pomonis et al., 2003; Rigoni et al., 2003). Protons act as endogenous activators and modulators of VR1 responses. Low pH enhances the apparent VR1-binding affinity of capsaicin, and potentiates the channel gating of VR1 receptors (Caterina et al., 1997; 2000; Tominaga et al., 1998; Olah et al., 2001; Ryu et al., 2003). Since inflammation leads to acidification of the inflamed tissue, VR1 is thought to play a major role in the transduction of inflammatory pain. As VR1 and TRPM8 are distantly related, and no antagonists have been described for TRPM8, we tested the effects of VR1 antagonists on TRPM8. Further, we investigated whether the responses of VR1 and TRPM8 towards agonists are influenced by pH.